The present invention is directed to a process for detection or identification of target moieties or target cells or from a cell sample.
Fluorescent dyes conjugated to one or more antibodies are commonly used for immunofluorescence analysis. A vast number of variants in view of antibodies, fluorescent dyes, flow cytometers, flow sorters, and fluorescence microscopes has been developed in the last two decades to enable specific detection and isolation of target cells. One issue in immunofluorescence technology is the detection threshold of the fluorescence emission, which can be enhanced, for example, by better detectors, filter systems, lasers, or modified fluorescent dyes i.e. with better quantum yield.
Although fluorescent labelling has found widespread use, it is by no means the only way of labelling and detecting cells. Besides fluorescent dyes other detection moieties have found use, such as transition metal isotope mass tag labelling or radioactive isotope labelling. Detection of labels is then performed with mass cytometry or scintillation assays.
Isolating target cells with flow sorting using fluorochrome-conjugates targeting the antigen of interest results in highly purified target cell fractions. One disadvantage is that after the sorting process cells are still labeled with the fluorochrome-conjugate used for the detection and identification during the sorting process.
As described for the isolation of antigen-specific T-cells with MHC-Multimers, disposition of labeling may affect target cells (U.S. Pat. No. 7,776,562). To avoid alterations of the cells, a release of fluorescent moiety and antigen recognizing moiety is possible after detection and/or sorting. U.S. Pat. No. 7,776,562 discloses a method of reversible fluorescent labeling based on indirect, non-covalent labeling of target cells with reversible peptide/MHC-Multimers or Fab-Streptamers. Low-affinity peptide/MHC- or Fab-monomers with StreptagII are multimerized via streptactin to provide complexes with high avidity for the target antigen. Reversibility of multimerization and monomerization is initiated by addition of the competitor Biotin. After dissociation of low-affinity peptide/MHC- or Fab-monomers the antigen is released from the interaction partners. Beside StreptagII/Streptactin-interaction another indirect binding interaction is described for reversible magnetic cell separation based on a PEO-Biotin/anti-Biotin-antibody interaction (EP2725359 A1).
WO20080918100 discloses a conjugate wherein an enzyme acts as activator for a fluorescent dye, which can be released from the target cells by radiation induced cleaving of the enzyme.
Multiple parameter labeling can be used to define and target more than one cell population or a specific subpopulation. However, depending on the respective equilibrium constant, a target cell labelled with a first antibody conjugate will be de-labelled after some time and re-labeled with either the first antibody conjugate again or with a second antibody conjugate. In the latter case, the information obtained will be a combination of the first and second labeling. Accordingly, simultaneous labelling with more than one antibody conjugate may reveal drawbacks as visualized in FIG. 1 and FIG. 2. FIG. 1 shows the mixture obtained by contacting two populations of cells (CD4 and CD8) with anti-CD8-PEO-Biotin/anti-Biotin-APC and anti-CD4-PEO-Biotin/anti-Biotin-PE. Due to the reversible nature of the Biotin/anti-Biotin complex, the resulting equilibrium comprises 4 instead of 2 cell-dye conjugates. The resulting equilibrium is determined by the kinetic and thermodynamic characteristic of the complexes and is reached after an exchange of the different binding partners leading to a fluorescent labeling of each target antigen with both fluorescent moieties. Instead of a specific labeling, a nearly unpredictable mixture is achieved. FIGS. 2a and b show two examples of this effect. The degree of exchange depends on parameters, like the selection of anti-Biotin-fluorochrome-conjugate as demonstrated in FIGS. 2a and b, as well as the concentration of the components of the complex. Therefore reversible fluorescent labeling based on reversible indirect systems is only suitable for single parameter and not for multiple parameter labeling. Compared to reversible indirect systems covalent conjugated antigen recognizing moiety and detection moiety provide specific labeling with the possibility to target multiple parameters. An example of a specific labeling of CD4+- and CD8+-target cells with covalently conjugated anti-CD4-PE and anti-CD8-APC is shown in FIG. 2c. 
Besides reversible systems based on specific competition of indirect, non-covalent binding interactions between antigen recognizing moiety and detection moiety, for magnetic cell separation several other reversible systems were developed. Known methods to release magnetic particles from target cells after separation are mechanical agitation or chemically or enzymatically cleavable linker to the magnetic particles. For example, U.S. Pat. No. 6,190,870 and WO 96/31776 discloses magnetic cell separation based on magnetic particles coated with dextran and/or linked via dextran to the antigen recognizing moiety. Subsequent cleavage of the isolated target cells from the magnetic particle is initiated by the addition of the dextran-degrading enzyme dextranase. Besides dextran-based magnetic particles the digestion of dextran-fluorochrome-conjugates is described in U.S. Pat. No. 5,719,031. In this case, the degree of labeling of dextran-fluorochrome-conjugates is high enough to furnish fluorescent quenching. Therefore degradation is accompanied by an enhancement of fluorescence emission signal, which is used for the quantification of the enzymatic digestion process.
Fluorescence quenching by connecting fluorescent dyes is also described in GB2372256. Cells are stained with a conjugate comprising a plurality of fluorescent dyes attached via a linker to an antibody. Since the high density of fluorescent dyes will quench the fluorescence signals, GB2372256 describes an enzymatic degradation of the linker in order to release fluorescent dyes from the conjugate. The released fluorescent dyes are not subject to self-quenching, resulting in more intense fluorescence signals, i.e. in better resolution. However, since the fluorescence signals are detected after release from the target, the identification of target moieties on the cell surface is not possible with the method according to GB2372256. Furthermore, it is not possible to detect more than one target simultaneously, since the resulting mix of fluorescence signals cannot be assigned to a specific conjugate and/or target.
Elimination of the fluorescence signal is essential for immunofluorescence technologies based on sequentially staining specimens. These technologies have been shown to provide a higher multiplexing potential compared to standard procedures using simultaneously labeling and detection. However, these technologies are based on oxidative destruction of conjugated fluorescent moieties by photo- or chemical bleaching procedures (U.S. Pat. No. 7,741,045 B2, EP 0810 428 B1 or DE10143757) and are subjected to steric hindrances by antibodies remaining on the specimen.
Besides chemical bleaching procedures to eliminate the fluorescence signal, DE 10143757 proposes the elimination of the fluorescence signal by enzymatic degradation of the antibodies. The enzymatic degradation of antibodies depends highly on the structure of the antibody and requires a specific selection of a “matching” pair of enzyme and antibody.